(1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteases, which generically consist of two parts - a peptidase and a chaperone-like ATPase. For several years, our studies focussed on the Clp proteases of E. coli, considered as a model system. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP to form active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then translocated axially into the digestion chamber inside ClpP. In FY07, we initiated a project to investigate the mobility of the N-terminal domains of ClpA that we had previously shown to undergo large fluctuations in the region distal to the hexameric ring of ATPase domains. The N-domains are connected to the ATPase domains by a flexible linker. Our working hypothesis is that shortening this linker will reduce the N-domain mobility, rendering them more visible in electron micrographs. Initial results with a 10- residue deletion mutant support this hypothesis. [unreadable] [unreadable] The proteosome is the machine responsible for ubiquitin-tagged protein degradation in eukaryotes. The 26S proteasome is composed by the 20s proteolytic chamber and the 19S complex which is involved in substrate binding and translocation into the 20s. The 19S complex comprises multiple regulatory proteins. We have begun to work on two of these, called rpn1 and rpn2. Both proteins lack ATPase activity and their functions are still unclear. A bioinformatics analysis has predicted that both proteins have alpha-solenoid folds, organized in a "croissant"-like shape. From negative staining EM and image averaging, we have obtained a representation of rpn2 that conforms to this expectation, and indicates four subdomains.[unreadable] [unreadable] (2a) Intracellular Trafficking: Interaction of Clathrin with Proteins that Regulate its Assembly. Clathrin plays a key role in intracellular trafficking, via its coating of membranous pits and vesicles (CCVs). Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. From these observations, we developed a model for uncoating. In FY07, we extended studies initiated during the previous year in which cryo-electron tomography is used to study the structures of individual CCVs isolated from bovine brain. Their polyhedral coats surround vesicles carrying cargoes of various shapes and sizes, including neurotransmitters, receptors and viruses. The tomograms divide coated particles into two sub-populations: 19% have internal vesicles and are true CCVs, and 81% lack internal membranes and so are clathrin baskets (CBs). The CCVs range from 80 to 134 nm in diameter, with vesicles of 30 to 68 nm. The CBs range from 66 to 120 nm, with those smaller than 80 nm too small to enclose the smallest vesicle (30 nm). The coats may be described by the symmetry of their polygonal facets. While many small polyhedral forms are possible in theory, many of them are not observed, suggesting that these forms are energetically disfavored. The common feature of these forbidden small polyhedra is that they have vertices with high curvature, placing a limit on how much the triskelion is able to bend. The smallest particle observed is a 28 vertex tetrahedral form, while the smallest CCV has 38 vertices with a 30 nm vesicle. In CCVs, the coat can be coupled to the vesicle through various proteins, of which the bulk are adaptor proteins. In our tomograms we see density between the clathrin N-termini and the membrane, with many shapes and sizes consistent with known structures of adaptor proteins. The vesicle is always located off-center relative to the coat, possibly reflecting the polarity inherent in how these vesicles bud off during endocytosis. In many CVs, the membrane is actually in contact with the clathrin N-termini, suggesting that clathrin itself may be able to interact directly with the membrane.[unreadable] [unreadable] (2b) Intracellular Trafficking: The Retromer Cargo-recognition Complex. The retromer is required for numerous intracellular transactions, such as the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric Ig receptor, Wnt gradient formation, iron transporter recycling, and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes, the cargo-recognition complex and a membrane-targeting complex. We are participating in a study to define the structures of these subcomplexes, their interactions in the fully assembled retromer, and their interactions with targeted membranes. The cargo-recognition complex is a heterotrimer of three proteins, Vps26, Vps29 and Vps35. The crystal structure of a subcomplex of Vps29 and a C-terminal fragment of Vps35 shows that the latter molecule forms a horseshoe-shaped alpha-helical solenoid. From bioinformatic analysis, we infer that the same fold is observed through the rest of Vps35. This prediction is supported by electron microscopy and image processing of the intact Vps26-Vps29-Vps35 complex which is revealed as a somewhat flexible, filamentous structure, 21 nm long. A model that synthesizes all currently available data shows the alpha-solenoid extending along the full length of Vps35, with Vps26 bound at the opposite end from Vps29. This elongated structure presents multiple binding sites for the membrane-targeting complex and receptor cargo.